An osmotic model for the fusion of biological membranes

A molecular model for fusion-fission reactions in membranes is proposed that is based on data from studies on artificially induced cell fusion and on the behaviour of phospholipid bilayers: it is put forward as a framework for further investigations into this fundamental property of biological systems.     

Fluorescence decay kinetics of chlorophyll in photosynthetic membranes

The absorption of light by the pigments of photosynthetic organisms results in electronic excitation that provides the energy to drive the energy-storing light reactions. A small fraction of this excitation gives rise to fluorescence emission, which serves as a sensitive probe of the energetics and kinetics of the excited states. The wavelength dependence of the excitation and emission spectra can be used to characterize the nature of the absorbing and fluorescing molecules and to monitor the process of sensitization of the excitation transfer from one pigment to another. This excitation transfer process can also be followed by the progressive depolarization of the emitted radiation. Using time-resolved fluorescence rise and decay kinetics, measurements of these processes can now be characterized to as short as a few picoseconds. Typically, excitation transfer among the antenna or light harvesting pigments occurs within 100 psec, whereupon the excitation has reached a photosynthetic reaction center capable of initiating electron transport. When this trap is functional and capable of charge separation, the fluorescence intensity is quenched and only rapidly decaying kinetic components resulting from the loss of excitation in transit in the antenna pigment bed are observed. When the reaction centers are blocked or saturated by high light intensities, the photochemical quenching is relieved, the fluorescence intensity rises severalfold, and an additional slower decay component appears and eventually dominates the decay kinetics. This slower (1-2 nsec) decay results from initial charge separation followed by recombination in the blocked reaction centers and repopulation of the excited electronic state, leading to a rapid delayed fluorescence component that is the origin of variable fluorescence. Recent growth in the literature in this area is reviewed here, with an emphasis on new information obtained on excitation transfer, trapping, and communication between different portions of the photosynthetic membranes.     

Fluorescence dequenching kinetics of single cell-cell fusion complexes.

In an earlier paper which models the cell-cell (or virus-cell) fusion complex as two partial spherical vesicles joined at a narrow neck (Rubin, R. J., and Yi-der Chen. 1990. Biophys. J. 58:1157-1167), the redistribution by diffusion of lipid-like molecules through the neck between the two fused cell surfaces was studied. In this paper, we extend the study to the calculation of the kinetics of fluorescence increase in a single fusion complex when the lipid-like molecules are fluorescent and self-quenching. The formalism developed in this paper is useful in deducing fusion activation mechanisms from cuvette fluorescence measurements in cell-cell fusion systems. Two different procedures are presented: 1) an exact one which is based on the exact local density functions obtained from diffusion equations in our earlier study; and 2) an approximate one which is based on treating the kinetics of transfer of probes between the two fused cells as a two-state chemical reaction. For typical cell-cell fusion complexes, the fluorescence dequencing curves calculated from the exact and approximate procedures are very similar. Due to its simplicity, the approximate method should be very useful in future applications. The formalism is applied to a typical cell-cell fusion complex to study the sensitivity of dequenching curves to changes in various fusion parameters, such as the radii of the cells, the radius of the pore at the fusion junction, and the number of probes initially loaded to the complex.     

Stochastic model of domain kinetics in biological membranes

A stochastic storage model based on the behavior of macroscopic variables of the system is used to describe the kinetics of raft-like domains in biological membranes. For a simple output model, we examine the features of the system behavior corresponding to the noise-induced nonequilibrium phase transitions. Characteristics of the behavior of the statistical system are obtained: an explicit form of the stationary distribution of the number of domains; ratios for the phase transition points; the expression for the first two moments of the random domain concentration, and the expression for the lifetime of membrane domains in the stationary state.     

Broadband method for measuring the ultrasonic absorption spectrum of biological tissues

An apparatus is described which in the frequency range from about 5 to 500 MHz allows the ultrasonic absorption coefficient of biological tissues to be measured with 1% accuracy. This apparatus is based on a pulse transmission method in which a computer-controlled mode of operation is combined with a RF substitution technique. Superheterodyne detection of the transmitted signal, multiple data recording and signal averaging result in a high sensitivity of the measuring method. The apparatus can be also used to analyze the ultrasonic pulse reflected by the sample.     

A simple method for measuring the kinetics of the formation of oxygen radicals

Sodium 2, 6-dichloroindophenol (DCIP Na ) was used to measure the kinetics of the formation of oxygen radicals. The oxygen radicals react with DCIP - , resulting in a decrease in DCIP concentration which is monitored by the decrease in A . A method based on this principle was demonstrated with xanthine/xanthine oxidase giving a K = 4.9x10 M. Cu significantly inhibited the enzyme reaction.     

Stoichiometry and kinetics of transport vesicle fusion with Golgi membranes

The in vitro complementation assay established by Rothman and co-workers continues to be an important tool to study intra-Golgi transport. In this study, kinetic modeling is used to identify four main parameters that, together, explain the basic features of an assay that is a modification of the original assay. First, the assay signal depends on the ratio of Golgi membranes to transport intermediates in the assay. Secondly, an inactivation rate describes how the activity of transport intermediates decreases over time. Thirdly, the rate at which transport intermediates irreversibly bind to Golgi membranes is measured independently of membrane fusion, thus allowing a quantitative distinction between these two steps. Fourthly, a single rate constant describes the remaining reactions, which result in membrane fusion. This approach of kinetic modeling of experiments is generally applicable to other in vitro assays of cell biological phenomena, permitting quantitative interpretations and an increased resolution of the experiments.     

Automated instrument for measuring biological transport kinetics over intervals of a few seconds

Automated sampling device was designed to permit determination of rates of biological transport of metabolites into cells. The substrate was automatically introduced into a stirred cell suspension at 37°C. The first sample was automatically taken after a mixing interval of 1 sec and nine subsequent samples were taken at programmable intervals (1 to 100 sec). The samples were forced by pressure differential (vacuum) through 0.4 μ pore size membranes and approximately 50 μl were collected in disposable cups. The duration of the sampling interval was controllable down to 0.1 sec. The samples preserved records of the substrate concentrations in solution at the time of filtration. With the use of suitable radioactive labeled isotopes, the changes in substrate concentrations may be conveniently measured by liquid scintillation spectrometry, but other analytical procedures of suitable sensitivity may be used. Initial and steady-state transport rates of succinate and glucose in Escherichia coli were obtained using the device.     

A novel micropipet method for measuring the bending modulus of vesicle membranes.

A theoretical model and an experiment are presented for determining the bending modulus of a bilayer vesicle membrane. The vesicle is held with a pipet having a radius between 1 and 2 microns, and the tension in the membrane is changed by changing the suction pressure. Then the vesicle membrane is deformed by aspirating it into a smaller pipet having a radius on the order of 0.5 microns. The relationship between the suction pressures in the two pipets is found to be linear, as predicted by the theoretical model. The curvature of the vesicle membrane at the pipet orifice and the bending modulus are found with the help of the model from the slope and the intercept of the linear experimental relationship between the suction pressures in the two pipets. The bending modulus for the two SOPC membranes studied in these experiments was found to be either 0.6 or 1.15 x 10(-19) J, which is similar to the values measured previously.     

Cholesterol sulfate inhibits the fusion of Sendai virus to biological and model membranes

Cholesterol sulfate is a component of several biological membranes. In erythrocytes, cholesterol sulfate inhibits hypotonic hemolysis, while in sperm, it can decrease fertilization efficiency. We have found cholesterol sulfate to be a potent inhibitor of Sendai virus fusion to both human erythrocyte and liposomal membranes. Cholesterol sulfate also raises the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine as demonstrated by differential scanning calorimetry and 31P nuclear magnetic resonance spectrometry. Although hexagonal phase structures are not readily found in biological membranes, there is a correlation between the effects of membrane additives on bilayer/non-bilayer equilibria and membrane stabilization. It is proposed that the ability of cholesterol sulfate to alter the physical properties of membranes contributes to its stabilization of biological membranes and the inhibition of membrane fusion.     

The influence of surface charge on the kinetics of ion-translocation across biological membranes

Rate equations have been developed which describe the concentration dependence for ion-translocation across charged membranes for those cases in which the translocation process can be considered to be formally equivalent with an enzymic process of a Michaelis-Menten type. We have limited ourselves to those cases in which the ion-translocational step through the membrane is electroneutral. In addition it is assumed that the sites on the membrane involved in the ion-translocation process can not move through the membrane when these sites are not occupied by ions.It is shown that in general deviations from Michaelis-Menten kinetics may be expected. In case of monovalent ion-translocation across oppositely charged membranes apparent negative homotrope cooperative effects may occur, whereas for ion-translocation across equally charged membranes apparent positive homotrope cooperative effects may be found. When the bulk aqueous phase also contains polyvalent ions both types of effects may occur both in the case of ion-translocation across oppositely charged membranes as well as with ion-translocation across a membrane of which the sign of the surface charge is the same as that of the ion translocated.Under limited conditions, also apparent single Michaelis-Menten kinetics may be observed. In these cases, however, the apparent K_m generally is no linear function of the concentration of a competing ion. It is shown that even when an ion does not bind to the translocation sites the K_m is affected by increasing concentrations of this ion, a phenomenon which is not expected when the membrane is not charged. The effects of divalent ions, added to the bulk aqueous phase as 1-1-electrolytes, upon the K_m are discussed in connection with in literature reported effects of Ca⁺⁺ upon the rate of uptake of several monovalent ions into plant cells.     

Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes

A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion of Sendai virions with phosphatidylcholine-cholesterol liposomes reflects the viral activity required for fusion with biological membranes

Sendai virus envelopes were reconstituted after solubilization of intact virions with either Triton X-100 or octylglucoside. Envelopes obtained from Triton X-100, but not from octylglucoside solubilized virions, were hemolytic and promoted cell-cell fusion. Fluorescence dequenching studies [using N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine-bearing viral envelopes] revealed that both preparations fused with negatively charged phospholipids. Fusion with phosphatidylcholine (PC)/cholesterol (chol) liposomes was promoted only by the hemolytic viral envelopes. Fluorescence dequenching studies, using intact virions bearing octadecylrhodamine B chloride, revealed that intact virions fused with PC/chol as well as with negatively charged phospholipids. Only fusion with PC/chol liposomes was inhibited by phenylmethylsulfonyl fluoride and dithiothreitol, reagents which are known to block the viral ability to fuse with biological membranes.     

Identification of nonlinear models of biological membranes using the voltage-clamp method

The method for identification of nonlinear systems proposed in 1952 by Hodgkin and Huxley is mathematically justified. A procedure for the application of this method is developed, including the development of the structure of a mathematical model, carrying out a series of tests with special chosen signals, and determination of unknown parameters. Basic requirements for the admissible sets of input and output signals and to the system operator have been determined. It is shown that this operator should be totally continuous and that the minimum number of unknown parameters and the minimum complexity of the operator structure should give an approximation of the necessary quality. The pros and cons of the Hodgkin-Huxley and Noble mathematical models and the methods used for their development are discussed. A structure for the operator for the identification of mathematical models of excitable membranes with a large number of membrane currents is proposed. It is found that the nonlinear electrical properties of biological membranes can be identified using tests with other types of “clamped” parameters, such as the current, ramp voltage, etc.     

GTP stimulates fusion between homologous and heterologous nuclear membranes

The tissue and species specificity of GTP-stimulated nuclear membrane fusion has been examined. The fusion capacity of the membranes of nuclei isolated from two different tissue sources and three different animal species was determined. In all cases the incubation of isolated nuclei in the presence of 0.5 mM GTP led to the pairing of nuclei and formation of continuous outer membranes between the nuclei as a result of membrane fusion. Experiments using mixtures of nuclei from the different sources demonstrated that hybrid nuclear membranes could be formed as a result of the fusion between the outer membranes of heterologous nuclear pairs. The results suggest that the capacity for nuclear membranes to fuse in the presence of GTP is highly conserved when viewed on an evolutionary basis.